A non-invasive method to produce pressure ulcers of varying severity in a spinal cord-injured rat model.

STUDY DESIGN: Experimental study. OBJECTIVES: The objective of this study was to establish a non-invasive model to produce pressure ulcers of varying severity in animals with spinal cord injury (SCI). SETTING: The study was conducted at the Johns Hopkins Hospital in Baltimore, Maryland, USA. METHODS: A mid-thoracic (T7-T9) left hemisection was performed on Sprague-Dawley rats. At 7 days post SCI, rats received varying degrees of pressure on the left posterior thigh region. Laser Doppler Flowmetry was used to record blood flow. Animals were killed 12 days after SCI. A cardiac puncture was performed for blood chemistry, and full-thickness tissue was harvested for histology. RESULTS: Doppler blood flow after SCI prior to pressure application was 237.808±16.175 PFUs at day 7. Following pressure application, there was a statistically significant decrease in blood flow in all pressure-applied groups in comparison with controls with a mean perfusion of 118.361±18.223 (P<0.001). White blood cell counts and creatine kinase for each group were statistically significant from the control group (P=0.0107 and P=0.0028, respectively). CONCLUSIONS: We have created a novel animal model of pressure ulcer formation in the setting of a SCI. Histological analysis revealed different stages of injury corresponding to the amount of pressure the animals were exposed to with decreased blood flow immediately after the insult along with a subsequent marked increase in blood flow the next day, conducive to an ischemia-reperfusion injury (IRI) and a possible inflammatory response following tissue injury. Following ischemia and hypoxia secondary to microcirculation impairment, free radicals generate lipid peroxidation, leading to ischemic tissue damage. Future studies should be aimed at measuring free radicals during this period of increased blood flow, following tissue ischemia.

Combination of HIF-1α gene transfection and HIF-1-activated bone marrow-derived angiogenic cell infusion improves burn wound healing in aged mice.

Impaired burn wound healing in the elderly represents a major clinical problem. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In a murine burn wound model, aged mice had impaired wound healing associated with reduced levels of HIF-1. When gene therapy with HIF-1 alone did not correct these deficits, we explored the potential benefit of HIF-1 gene therapy combined with the intravenous infusion of bone marrow-derived angiogenic cells (BMDACs) cultured with dimethyloxalylglycine (DMOG). DMOG is known to reduce oxidative degradation of HIF-1. The mice treated with a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α)+BMDACs had more rapid wound closure. By day 17, there were more mice with completely closed wounds in the treated group (χ(2), P=0.05). The dermal blood flow measured by laser Doppler showed significantly increased wound perfusion on day 11. Homing of BMDACs to the burn wound was dramatically enhanced by CA5-HIF-1α gene therapy. HIF-1α mRNA expression in the burn wound was increased after transfection with CA5-HIF-1α plasmid. Our findings offer insight into the pathophysiology of burns in the elderly and point to potential targets for developing new therapeutic strategies.

Hindbrain GABA receptors influence parasympathetic outflow to the stomach.

Blockade of gamma-aminobutyric acid receptor function by direct microinjection of bicuculline into the nucleus ambiguous in cats produced a marked increase in gastric motility which was mediated by the vagus nerve. This effect was reversed by muscimol. These data indicate that the nucleus ambiguous may be an important brain site influencing gastric function and that the neurotransmitter controlling parasympathetic overflow from this nucleus to the stomach is gamma-aminobutyric acid.

Ischemia reperfusion-facilitated sinusoidal endothelial cell injury in liver transplantation and the resulting impact of extravasated platelet aggregation.

BACKGROUND: The exact sequence of events leading to ultimate hepatocellular damage following ischemia/reperfusion (I/R) is incompletely understood. In this article, we review a mechanism of organ dysfunction after hepatic I/R or immunosuppressive treatment, in addition to the potential of liver sinusoidal endothelial cell (LSEC) protection and antiplatelet treatment for the suppression of hepatocellular damage. METHODS: A review of the literature, utilizing PubMed-NCBI, was used to provide information on the components necessary for the development of hepatocellular damage following I/R. RESULTS: It is well-established that LSECs damage following hepatic I/R or immunosuppressive treatment followed by extravasated platelet aggregation (EPA) is the root cause of organ dysfunction in liver transplantation. We have classified three phases, from LSECs damage to organ dysfunction, utilizing the predicted pathogenic mechanism of sinusoidal obstruction syndrome. The first phase is detachment of LSECs and sinusoidal wall destruction after LSECs injury by hepatic I/R or immunosuppressive treatment. The second phase is EPA, accomplished by sinusoidal wall destruction. The various growth factors, including thromboxane A2, serotonin, transforming growth factor-beta and plasminogen activator inhibitor-1, released by EPA in the Disse's space of zone three, induce portal hypertension and the progression of hepatic fibrosis. The third phase is organ dysfunction following portal hypertension, hepatic fibrosis, and suppressed liver regeneration through various growth factors secreted by EPA. CONCLUSION: We suggest that EPA in the space of Disse, initiated by LSECs damage due to hepatic I/R or immunosuppressive treatment, and activated platelets may primarily contribute to liver damage in liver transplantation. Endothelial protective therapy or antiplatelet treatment may be useful in the treatment of hepatic I/R following EPA.

Secretion of intrinsic factor from dispersed mucosal cells isolated from guinea pig and rabbit stomach.

In dispersed mucosal cells prepared from rabbit and guinea pig stomach, the secretion of intrinsic factor was constant (0.3-0.4%/min) for at least 30 min incubation at 37 degrees C. Histamine or isobutyl methylxanthine increased cyclic AMP and intrinsic factor secretion in both cells preparations. Isobutyl methylxanthine potentiated and cimetidine competitively inhibited (ki = 5.10-7 M) both effects of histamine. Dibutyryl cyclic AMP (1.0 mM), also caused a 3-fold increase in intrinsic factor secretion. These results suggest that in rabbit and guinea pig histamine interacts with H2-receptors to increase cyclic AMP which mediates the rise in the rate of intrinsic factor secretion.

Acidic fibroblast growth factor overexpression partially protects 3T3 fibroblasts from apoptosis induced by synthetic retinoid CD437.

Retinoids are proapoptotic compounds with therapeutic potential for treating cancer. We evaluated the apoptotic effect of the novel retinoid CD437, and particularly its relationship to Akt and acidic fibroblast growth factor (aFGF). We hypothesized that the novel synthetic retinoid CD437 would exert its apoptotic effect by reducing the activity of Akt. We further hypothesized that aFGF would protect against CD437 apoptosis by preserving the activity of Akt. Initially we demonstrated that CD437 produces apoptotic cell death in NIH 3T3 fibroblasts, and that this effect is attenuated in fibroblasts transfected to express aFGF. Next we assessed Akt activity and showed that phospho-Akt is significantly reduced in 3T3 cells exposed to CD437. We showed that this effect is less pronounced in aFGF transfected 3T3 cells. Furthermore, we observed that the addition of exogenous aFGF to 3T3 cells significantly increases Akt phosphorylation. These findings tend to confirm our hypothesis that reduction in Akt activation is a mechanism involved in the apoptotic effect of the retinoid CD437, and that preservation of Akt phosphorylation occurs in response to aFGF and appears to explain the partially protective effect of aFGF for 3T3 cells vis a vis CD437.

Alterations in serum creatine kinase and lactate dehydrogenase. Association with abdominal aortic surgery, myocardial infarction and bowel necrosis.

Experimental studies have shown that peripheral serum creatine kinase and lactate dehydrogenase change with bowel infarction. Some clinical reports have suggested that similar changes occur in patients. This prospective study documents the changes in these enzymes associated with acute myocardial infarction, acute bowel necrosis (MES INF), and uncomplicated abdominal aortic reconstruction. Analysis of 15 patients with AMI, 13 patients undergoing major AAS, and eight patients with MES INF has shown that these conditions may be differentiated by analysis of serum CK and LD isoenzymes. The study suggests that in the absence of electrocardiographic changes, a patient with epigastric distress with elevated levels of serum CK and either CK-MB or CK-BB bands present may well have a mesenteric rather than a myocardial infarction. Acute myocardial infarction can be ruled out further through analysis of serum LD1/LD2 ratios.

Taurodeoxycholate modulates the effects of pepsin and trypsin in experimental esophagitis.

Pepsin and trypsin cause erosive, hemorrhagic lesions in our rabbit model of experimental esophagitis. Since the gastroduodenal contents of patients with reflux esophagitis may also contain bile salts, we used our model to determine the effect that a bile salt, taurodeoxycholate (TDC), would have on the esophageal mucosa when combined with either pepsin in an acid perfusate (pH 2) or trypsin in an alkaline perfusate (pH 7.5). Indexes of esophageal injury included gross appearance of the mucosa, microscopic examination, and mucosal barrier integrity as determined by permeability to hydrogen ion. We found that when 5 mM TDC was combined with pepsin (0.3 mg/ml), the gross and microscopic changes of esophagitis, as well as net hydrogen ion flux, were diminished when compared with those observed with pepsin exposure alone. When increasing concentrations of TDC (2 to 10 mM) were added to pepsin, the morphologic degree of injury as well as hydrogen ion flux decreased in a dose-dependent manner. In contrast, when 5 mM TDC was combined with trypsin (1000 U/ml) in the alkaline perfusate, the gross and microscopic changes of esophagitis and the net of hydrogen ion flux were increased when compared with either bile salt or trypsin alone. These effects were also dose dependent. These data demonstrate that bile salts present in the gastroduodenal contents of patients with reflux esophagitis have the capacity to modulate the effects of pepsin and trypsin on the esophageal mucosa.

Esophageal blood flow in the rabbit: response to calcium channel blockers.

Calcium channel blockers have recently been added to the therapeutic regimen for patients who have chest pain of esophageal origin. Although relief of symptoms has been reported, this has not always been associated with changes in esophageal contraction pressures or luminal pH. Myoischemia has been proposed as one possible mechanism for esophageal chest pain. We have investigated the effect of the calcium channel blockers verapamil, nifedipine, and diltiazem on esophageal blood flow in the rabbit model. Esophageal blood flow was measured three times in each rabbit with use of the radiolabeled microsphere technique after a 30-minute continuous infusion of (1) saline solution (baseline), (2) a low dose, and (3) a high dose of each agent. Esophageal mucosal blood flow significantly decreased with nifedipine but was unchanged with verapamil and diltiazem. Esophageal muscle blood flow significantly increased--approximately 100% after administration of each of the calcium channel blockers. Thus esophageal muscle blood flow is enhanced after administration of calcium channel blockers, and this may be one therapeutic mechanism of the calcium channel blockers in the relief of esophageal chest pain in some esophageal diseases.

Role of the components of the gastroduodenal contents in experimental acid esophagitis.

Esophagitis has been associated with the reflux of acidic gastroduodenal contents. These contents may contain not only HCl but also pepsin, bile, and pancreatic enzymes. This experiment was designed to compare the roles of these components in experimental acid esophagitis. The esophagus of the rabbit was cannulated and perfused continuously via a recirculating system with pH 2 acid test solution. Net flux of H+, K+, glucose, and hemoglobin plus the recovery of tritiated water were determined before and after the addition of pepsin, taurodeoxycholate, or trypsin. Afterward the esophageal segments were graded for gross and microscopic esophagitis. These studies show that pepsin caused significant gross and microscopic esophagitis. Moreover, pepsin also caused significant increases in H+, K+, glucose, and hemoglobin flux as well as decreased recovery of tritiated water. Taurodeoxycholate increased esophageal mucosal permeability to H+, K+, and glucose and decreased the recovery of tritiated water but did not cause significant pathologic change. Trypsin and acid alone did not result in significant esophagitis by either pathologic or ionic permeability criteria. These results show tha disruption of the esophageal mucosal barrier cannot be equated with pathologic injury and that different components of the gastroduodenal contents may have different sites or mechanisms of injury.

H+ back diffusion interferes with intrinsic reactive regulation of esophageal mucosal blood flow.

The esophageal mucosa maintains a barrier that is relatively impermeable to glucose, H+, and other small molecules. Injury of the mucosa causes disruption of this barrier, manifest initially by increased permeability to small molecules. In the stomach the mucosa is protected from gross ulceration in the presence of bile-induced H+ back diffusion (JH+) by increases in mucosal blood flow (Qm). Qm to the esophagus during injury has never been studied. We explored the possibility that esophageal Qm would increase as a compensatory reaction to early barrier disruption. Rabbits (2 to 4 kg) were anesthetized and the in situ esophagus was luminally perfused for two 1-hour periods with subulcerogenic concentrations of bile salts, pepsin, or trypsin in the presence (pH 2) or absence (pH 7) of acid. Qm was measured with 15 mu radioactive microspheres in nine experimental groups with a total of 62 rabbits. Changes in Qm were compared with changes in permeability of the esophageal barrier to glucose, Na+, and H+. When the mucosal barrier was broken by bile salts or trypsin at a neutral pH, no acid back diffusion occurred and barrier disruption was accompanied by dramatic increases in esophageal mucosal blood flow. In contrast, barrier disruption by bile salts, pepsin, or acid during pH 2 perfusions failed to elicit increases in Qm when significant JH+ (50 microEq/hr) occurred. These results demonstrate a loss in reactive regulation of esophageal Qm in the presence of significant JH+ that may contribute to the injury seen in acid reflux esophagitis.

Insulin-like growth factor I receptors in the arteries of the rabbit: autoradiographic mapping and receptor characterization.

Insulin-like growth factor I (IGF-I) is a polypeptide hormone structurally related to insulin with insulin-like metabolic effects. It is a potent mitogen, eliciting cell multiplication in tissue culture by increasing deoxyribonucleic acid and protein synthesis. IGF-I was found to promote the growth of cultured arterial smooth muscle cells. We studied the in situ distribution of IGF-I receptors in different arteries of the rabbit by autoradiography and examined their binding characteristics in the wall of the thoracic aorta. The thoracic and abdominal aortas and carotid, superior mesenteric, renal, and iliac arteries of three adult New Zealand rabbits were harvested and stored at -70 degrees C. Autoradiographic analysis of 125I-labeled IGF-I binding to frozen arterial sections showed that silver-grain density was consistently located in the arterial wall. Binding studies in the thoracic aorta demonstrated high-affinity IGF-I receptors with a dissociation constant of 2 nmol/L and maximum IGF-I binding capacity of 4.17 pmol/mg protein. Inhibition studies with insulin, IGF-I, and IGF-II showed that these binding sites were more specific for IGF-I than for IGF-II or insulin, with a concentration of peptide that inhibits 50% of maximum binding of 1.75 nmol/L, 5 nmol/L, and greater than 100 mumol/L, respectively. The presence of high-affinity, specific IGF-I receptor binding in rabbit arteries suggests that IGF-I plays an important role in regulating the multiplication of arterial smooth muscle cells; a role that may prove important in different pathologic processes.

Capsaicin-sensitive nerves mediate esophageal mucosal protection.

The esophageal mucosa is exposed to damaging agents both by ingestion and reflux. Using our in vivo rabbit model of esophagitis, we have observed that acute luminal exposure (within 1 to 5 minutes) to potentially harmful agents, such as acid, bile, or ethanol, induces a rapid increase in mucosal blood flow; whereas prolonged exposure (10 to 60 minutes) results in mucosal injury and ablation of blood flow. We have also shown that capsaicin-sensitive mucosal afferent nerves can modulate esophageal blood flow. These findings led us to hypothesize that the reactive increase in blood flow induced by luminal agents represents a mechanism of protection mediated by capsaicin-sensitive nerves. The objective of these experiments was to determine if luminal capsaicin, a specific probe for visceral afferent nerves, could both preserve mucosal blood flow and protect against ethanol injury. Rabbits were subjected to luminal instillation of 50% ethanol with or without 1% capsaicin. Blood flow was measured with microspheres at baseline and after 2 and 10 minutes. Rabbits exposed only to ethanol developed severe mucosal injury coincident with near ablation of mucosal blood flow. In contrast, rabbits exposed to ethanol with capsaicin showed protection of the epithelium with a sixfold increase in mucosal blood flow. We conclude that capsaicin-sensitive nerves in the esophagus are local effectors of mucosal protection by virtue of preserving blood flow.

Parathyroidectomy promotes wound healing and prolongs survival in patients with calciphylaxis from secondary hyperparathyroidism.

BACKGROUND: Calciphylaxis is a rare but life-threatening condition occasionally affecting patients with secondary hyperparathyroidism. Parathyroidectomy has been advocated as the only potentially curative intervention. METHODS: Between January 1989 and May 2000, 13 patients with pathologic/clinical criteria of calciphylaxis were treated at our institution. Of these 13 patients, 7 were managed with medical therapy alone, and 6 were referred for parathyroidectomy. The medical records were reviewed, and patients/relatives were interviewed. RESULTS: All patients had cutaneous wounds requiring local debridement predominantly located on the lower extremities or abdominal wall. Six patients underwent subtotal (3.5 gland) parathyroidectomy without morbidity. All 6 had significant reductions in parathyroid hormone levels after surgery (mean decrease, 94% +/- 0%), and all reported resolution of pain and healing of cutaneous wounds. Of the remaining 7 patients who had medical management alone, 5 eventually died of complications related to calciphylaxis. In comparing the 2 groups, patients who underwent parathyroidectomy had a significantly longer median survival than those who did not have surgery (36 vs 3 months, P =.021). CONCLUSIONS: Calciphylaxis frequently causes gangrene, sepsis, and eventual death. Parathyroidectomy can be performed with minimal morbidity and is associated with resolution of pain, wound healing, and a significantly longer median survival. Therefore, patients with secondary hyperparathyroidism and signs/symptoms of calciphylaxis should be referred promptly for consideration of parathyroidectomy.

Reversibility of deoxycholate-induced cellular hypercalcemia in rabbit gastric mucosal cells.

BACKGROUND: Bile acid exposure produces cellular hypercalcemia in gastric and hepatic cells. It is not known, however, whether this event contributes to cell injury or if it results from passive equilibration of calcium ion concentrations across the membranes of irreversibly damaged cells. This study was performed to determine whether the cellular hypercalcemia produced by bile acid exposure in gastric cells is reversible and to determine whether the source of this hypercalcemia is from intracellular stores of calcium, extracellular sources, or both. METHODS: Cytosolic free calcium concentrations ([Ca]i) were measured in rabbit gastric mucosal cells that had been loaded with the intracellular probe FURA-2. Measurements were performed in suspensions of dispersed cells by using standard spectrofluorometry and in primarily cultured cells by using fluorescence videomicroscopy. Measurements were made before and after exposure to 0.2, 0.5, and 1.0 mmol/L deoxycholic acid (DC). These measurements were made in the presence of 1 mmol/L extracellular calcium and in the absence of any extracellular calcium (0.5 mmol/L EGTA). RESULTS: In experiments with dispersed cells and spectrofluorometry, [Ca]i increased from a pretreatment level of 194 +/- 8 nmol/L to 396 +/- 21 nmol/L within 3 minutes of exposure to 0.2 mmol/L DC. When these cells were washed and resuspended in DC-free medium, [Ca]i] decreased to 180 +/- 5 nmol/L. In experiments with cultured cells and fluorescence videomicroscopy, rapid, reversible hypercalcemia was observed after exposure to 0.5 and 1.0 mmol/L DC. Removal of extracellular calcium from the incubating medium reduced both the magnitude and duration of the observed hypercalcemia. CONCLUSIONS: These data show that the cellular hypercalcemia that accompanies DC-induced injury in gastric cells is a reversible event. The initial increase in [Ca]i appears to come from both intracellular and extracellular sources, although sustained hypercalcemia requires a source of extracellular calcium. As a reversible event, cellular hypercalcemia may be an important pathophysiologic feature of bile acid induced injury of the upper gastrointestinal tract.

Paraduodenal hernia: diagnosis and surgical management.

Paraduodenal hernia is an unusual cause of intestinal obstruction, but one with which all surgeons should be familiar. We reviewed the anatomy, pathophysiology, initial symptoms, radiographic criteria for diagnosis, and subsequent therapy of five patients treated for paraduodenal hernia at Walter Reed Army Medical Center. Contrast radiography of the small intestine remains the mainstay of preoperative diagnosis. Essential components of treatment include bowel reduction and obliteration of the hernia defect by simple closure or by wide opening of the sac. Further recommendations include sparing the inferior mesenteric vessels during the repair of left paraduodenal hernias and transpositioning the right colon to the left side of the abdomen for repair of right paraduodenal hernias.

Esophageal mucosal blood flow: a central role for calcitonin gene-related peptide.

BACKGROUND: Esophageal mucosal blood flow is a dynamic phenomenon dependent on luminal content. Reactive hyperemia, likely a factor in mucosal protection, follows luminal exposure to noxious substances, including bile. The mediators of this response are unknown, although the likelihood is that visceral afferent nerves play a major role. The purpose of this study was to determine whether substance P, calcitonin gene-related peptide (CGRP), or adenosine could mediate this reactive blood flow response. METHODS: Esophageal mucosal blood flow was studied in a rabbit model with the radiolabeled microsphere technique. The effect of intraarterial infusion of CGRP and substance P and intravenous adenosine was studied. Subsequently, the hyperemic response to luminal deoxycholate was measured in the presence of antagonists to CGRP, substance P, and adenosine. Immunohistochemical studies were performed to determine the distribution of CGRP and substance P in the esophagus. RESULTS: CGRP proved to be a potent stimulus to mucosal blood flow. The presence of a CGRP antagonist reduced mucosal blood flow at baseline and after exposure to deoxycholate. Antagonists to substance P and adenosine had no effect on baseline and deoxycholate-stimulated blood flow. CONCLUSIONS: CGRP is likely a major mediator involved in the regulation of esophageal mucosal blood flow.

Use of vascularized abdominal wall pedicle flaps to grow small bowel neomucosa.

Growing new mucosa from remnants of small bowel remaining in patients with short-bowel syndrome might offer a strategy for solving this clinical problem. We have performed a series of experiments investigating the possibility of growing rabbit ileal mucosa on vascularized pedicle flaps of abdominal wall musculature based on the inferior epigastric artery. By patching a defect of distal ileum with a skeletal muscle flap, we were able to demonstrate bowel augmentation by neomucosal ingrowth. Light and scanning electron microscopy confirmed the presence of essentially normal mucosa with well-developed villi atop the skeletal muscle pedicle flap. The mucosa was stripped from the skeletal muscle and compared with stripped mucosa from adjacent ileum in the Ussing chamber in 11 rabbits. The electrophysiologic studies showed no significant difference between normal mucosa and neomucosa in short-circuit current (Isc), potential difference or tissue conductance. The addition of 10 mM glucose resulted in similar unidirectional glucose flux and increase in Isc in both tissues. Bile salt absorption was also similar in both tissues. We conclude that neomucosa can be grown on flaps of skeletal muscle and is similar to normal mucosa by microscopic and electrophysiologic evaluation.

The excluded small-bowel segment. A source of complications after small-bowel bypass.

Two cases of obstruction of the bypassed small intestine after jejunoileal shunt for obesity are presented. These cases illustrate the possible failure of radiologic visualization of the obstructed bowel since no gas traverses this bowel, as well as two of the possible causes-internal herniation and volvulus. A third cause, intussusception of the blind loop into the colon, has been reported. Obstruction of the bypassed bowel demands surgical intervention and could lead to perforation and peritonitis if untreated. Its prevention involves the closure of all mesenteric defects at the original operation. Surgeons should be aware of the possibility of these conditions in any patient who has had a small-bowel bypass operation.